Methods of treating disease with levoketoconazole

ABSTRACT

Provided herein is a method of administering levoketoconazole, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein the subject is also being administered a multidrug and toxin extrusion transporter 1 (MATE1) substrate or an organic cation transporter 2 (OCT2) substrate.

This application is a continuation of International Application No.PCT/US2020/020644, filed Mar. 2, 2020, which claims the benefit ofpriority of U.S. Provisional Patent Application Ser. No. 62/813,399filed Mar. 4, 2019, the disclosures of which are incorporated byreference in their entireties for all purposes.

Endogenous Cushing's disease is a rare, serious, and potentially lethalendocrine disease caused by excessive exposure of organs to cortisol. Inabout 80% of patients, excessive secretion of adrenocorticotrophichormone (ACTH) causes Cushing's syndrome, most commonly via a pituitarycorticotropic adenoma and less often via an extrapituitary tumor(ectopic ACTH syndrome) or, in rare instances, by an ectopiccorticotropin-releasing hormone-secreting tumor. In the remaining 20% ofpatients, Cushing's syndrome is ACTH-independent and is caused by excesscortisol secretion by unilateral adrenocortical tumors, bilateraladrenal hyperplasia, or dysplasia.

Nizoral™ (ketoconazole) is approved in the US as an antifungal agent forcertain systemic and recalcitrant skin fungal infections. KetoconazoleHRA® contains ketoconazole and is registered in some countries outsidethe US as a treatment for Cushing's syndrome. Ketoconazole reduces orinhibits adrenal steroid production by inhibiting several adrenalsteroidogenic enzymes, including CYP17A1 (also known as 17α-hydroxylase)and CYP11B1 (also known as mitochondrial 11β-hydroxylase). A directeffect on ectopic ACTH has also been observed in vitro. The efficacy ofketoconazole in treating Cushing's syndrome has not been the subject ofa large prospective clinical trial, although it has been the subject ofseveral small open-label trials and larger retrospective case series.Ketoconazole reportedly normalizes hypercortisolism in 30-70% ofpatients and reduces complications of excessive cortisol, includingdiabetes and hypertension, and other signs and symptoms of Cushing'ssyndrome.

Ketoconazole, however, has several known risks, includinghepatotoxicity. Elevated levels of transaminases are common.Ketoconazole also potently inhibits several drug-metabolizing enzymes,including CYP3A4, with a potential for marked drug interactions,including those leading to increased risk for QT prolongation. Despitethe availability of two FDA-approved drugs, ketoconazole continues to becommonly used off-label for treating Cushing's syndrome in the US, andby some accounts is the most frequently prescribed medical therapy forCushing's syndrome, reflecting the persistent need unmet by approvedtherapies.

Levoketoconazole (Recorlev™, COR-003, 2S,4Rcis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxyl]phenyl]piperazine)is an investigational cortisol synthesis inhibitor. Levoketoconazole isthe 2S,4R enantiomer of ketoconazole. Nonclinical and clinical datasuggest that compared with the 2R,4S enantiomer of ketoconazole,levoketoconazole more potently inhibits cortisol synthesis, and reacheshigher plasma concentrations after ketoconazole dosing.

Provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the subject is also being        co-administered a therapeutically effective amount of a        multidrug and toxin extrusion transporter 1 (MATE1) substrate or        an organic cation transporter 2 (OCT2) substrate;    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme, and    -   wherein the therapeutically effective amount of the MATE1        substrate or OCT2 substrate, is reduced relative to a subject        who is not being administered levoketoconazole, or a        pharmaceutically acceptable salt thereof.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancersin a subject in need thereof, wherein the subject is beingco-administered a therapeutically effective amount of a multidrug andtoxin extrusion transporter 1 (MATE1) substrate or an organic cationtransporter 2 (OCT2) substrate, comprising:

-   -   reducing the amount of MATE1 substrate or OCT2 substrate being        administered to the subject, and    -   subsequently initiating administration of a therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme.

In some embodiments, reducing the amount of MATE1 substrate or OCT2substrate being administered to the subject comprises discontinuingadministration of the MATE1 substrate or OCT2 substrate.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the subject is also being        co-administered a therapeutically effective amount of metformin,        or a pharmaceutically acceptable salt thereof;    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme, and    -   wherein the therapeutically effective amount of metformin, or a        pharmaceutically acceptable salt thereof, is reduced relative to        a subject who is not being administered levoketoconazole, or a        pharmaceutically acceptable salt thereof.

In some embodiments, reducing the amount of metformin being administeredto the subject comprises discontinuing administration of the metformin.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancersin a subject in need thereof, wherein the subject is beingco-administered a therapeutically effective amount of metformin,comprising:

-   -   reducing the amount of metformin being administered to the        subject, and    -   subsequently initiating administration of a therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme.

Also provided is a method of treating a disease chosen from Cushing'sdisease, syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof, is determined via a titration scheme,        and    -   subsequently determining that the patient is to begin treatment        with a multidrug and toxin extrusion transporter 1 (MATE1)        substrate or an organic cation transporter 2 (OCT2) substrate,        wherein MATE1 substrate or OCT2 substrate, is administered in an        amount that is less than the amount that would be administered        to a patient who is not being administered levoketoconazole, or        a pharmaceutically acceptable salt thereof.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof, is determined via a titration scheme,        and    -   subsequently determining that the patient is to begin treatment        with metformin, wherein the metformin is administered in an        amount that is less than the amount that would be administered        to a patient who is not being administered levoketoconazole, or        a pharmaceutically acceptable salt thereof.

In some embodiments, the starting dose is less than the amount thatwould be administered to a patient who is not being administeredlevoketoconazole, or a pharmaceutically acceptable salt thereof. In someembodiments, the starting dose is increased by an incremental amount(e.g., 250 mg) that is less than the amount that would be used with apatient who is not being administered levoketoconazole, or apharmaceutically acceptable salt thereof (e.g., 500 mg).

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,in a subject in need thereof, wherein the subject is also beingadministered metformin, comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof,    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme, and    -   wherein the administration of levoketoconazole, or a        pharmaceutically acceptable salt thereof, increases the systemic        exposure to metformin by about 2-fold.

These and other aspects of the invention will be apparent upon referenceto the following detailed description. To this end, various referencesare set forth herein which describe in more detail certain backgroundinformation, procedures, compounds, and/or compositions, and are eachhereby incorporated by reference in their entirety.

DETAILED DESCRIPTION

When introducing elements of the present disclosure or the embodiment(s)thereof, the articles “a”, “an”, “the” and “said” are intended to meanthat there are one or more of the elements. The terms “comprising”,“including” and “having” are inclusive and mean that there may beadditional elements other than the listed elements.

The term “and/or” when in a list of two or more items, means that any ofthe listed items can be employed by itself or in combination with one ormore of the listed items. For example, the expression “A and/or B” meanseither or both of A and B, i.e. A alone, B alone or A and B incombination. The expression “A, B and/or C” is intended to mean A alone,B alone, C alone, A and B in combination, A and C in combination, B andC in combination or A, B, and C in combination.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” or “between n₁ . . . and n₂” is used, where n₁ and n₂ are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 μM(micromolar),” which is intended to include 1 μM, 3 μM, and everythingin between to any number of significant figures (e.g., 1.255 μM, 2.1 μM,2.9999 μM, etc.).

The term “about” qualifies the numerical values that it modifies,denoting such a value as variable within a margin of error. When nomargin of error, such as a standard deviation to a mean value given in achart or table of data, is recited, the term “about” means that rangewhich would encompass the recited value and the range which would beincluded by rounding up or down to that figure, considering significantfigures.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treating a disease or disorder or onthe effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. Treatment may also be preemptive in nature, i.e.,it may include prevention of disease. Prevention of a disease mayinvolve complete protection from disease, for example as in the case ofprevention of infection with a pathogen or may involve prevention ofdisease progression. For example, prevention of a disease may not meancomplete foreclosure of any effect related to the diseases at any level,but instead may mean prevention of the symptoms of a disease to aclinically significant or detectable level. Prevention of diseases mayalso mean prevention of progression of a disease to a later stage of thedisease. In certain embodiments, prevention of a disease may involveprevention of attacks of an intermittent nature, as well as preventionof a permanent state of muscle weakness, such as an irreversible stateof impairment owing to underlying disease.

The term “patient” is generally synonymous with the term “subject” andincludes all mammals including humans. Examples of patients includehumans, livestock such as cows, goats, sheep, pigs, and rabbits, andcompanion animals such as dogs, cats, rabbits, and horses. Preferably,the patient is a human.

As used herein, a patient is said to “tolerate” a dose of a compound ifadministering that dose to that patient does not result in anunacceptable adverse event or an unacceptable combination of adverseevents. One of skill in the art will appreciate that tolerance is asubjective measure and that what may be tolerable to one patient may notbe tolerable to a different patient. For example, one patient may not beable to tolerate headache, whereas a second patient may find headachetolerable but is not able to tolerate vomiting, whereas for a thirdpatient, either headache alone or vomiting alone is tolerable, but thepatient is not able to tolerate the combination of headache andvomiting, even if the severity of each is less than when experiencedalone.

As used herein, an “adverse event” is an untoward medical occurrenceassociated with treatment with a pharmaceutical agent.

As used herein, the term “hormone-sensitive cancer” refers to any cancerwhich may be affected by a hormone; hormones typically increaseproliferation of hormone-sensitive cancers.

As used herein, “up-titration” of a compound refers to increasing theamount of a compound to achieve a therapeutic effect that occurs beforedose-limiting intolerability for the patient. Up-titration can beachieved in one or more dose increments, which may be the same ordifferent.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present disclosure includes compounds listed above in theform of salts, including acid addition salts. Suitable salts includethose formed with both organic and inorganic acids. Such acid additionsalts will normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable.

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate (p-tosylate), and undecanoate. Also, basic groupsin the compounds disclosed herein can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.Examples of acids which can be employed to form therapeuticallyacceptable addition salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, and citric. Salts can also be formed by coordinationof the compounds with an alkali metal or alkaline earth ion. Hence, thepresent disclosure contemplates sodium, potassium, magnesium, andcalcium salts of the compounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

A salt of a compound can be made by reacting the appropriate compound inthe form of the free base with the appropriate acid.

Metformin refers to N,N-dimethylimidodicarbonimidic diamidehydrochloride. A formulation of metforminhas been previously reported inthe FDA approved drug label GLUCOPHAGE as an adjunct to diet andexercise to improve glycemic control in adults with type 2 diabetesmellitus.

GLUCOPHAGE tablets contain 500 mg, 850 mg, or 1000 mg of metforminhydrochloride, which is equivalent to 389.93 mg, 662.88 mg, 779.86 mgmetformin base, respectively. Each tablet contains the inactiveingredients povidone and magnesium stearate. In addition, the coatingfor the 500 mg and 850 mg tablets contains hypromellose and the coatingfor the 1000 mg tablet contains hypromellose and polyethylene glycol.GLUCOPHAGE XR contains 500 mg or 750 mg of metformin hydrochloride,which is equivalent to 389.93 mg, 584.90 mg metformin base,respectively. GLUCOPHAGE XR 500 mg tablets contain the inactiveingredients hypromellose, microcrystalline cellulose, sodiumcarboxymethyl cellulose, and magnesium stearate. GLUCOPHAGE XR 750 mgtablets contain the inactive ingredients hypromellose, sodiumcarboxymethyl cellulose, magnesium stearate and iron oxide pigment red.

The standard dosages for metformin are:

Adult Dosage for GLUCOPHAGE:

-   -   Starting dose: 500 mg orally twice a day or 850 mg once a day,        with meals    -   Increase the dose in increments of 500 mg weekly or 850 mg every        2 weeks, up to a maximum dose of 2550 mg per day, given in        divided doses    -   Doses above 2000 mg may be better tolerated given 3 times a day        with meals

Adult Dosage for GLUCOPHAGE XR:

-   -   Starting dose: 500 mg orally once daily with the evening meal    -   Increase the dose in increments of 500 mg weekly, up to a        maximum of 2000 mg once daily with the evening meal    -   Patients receiving GLUCOPHAGE may be switched to GLUCOPHAGE XR        once daily at the same total daily dose, up to 2000 mg once        daily

Pediatric Dosage for GLUCOPHAGE:

-   -   Starting dose: 500 mg orally twice a day, with meals    -   Increase dosage in increments of 500 mg weekly up to a maximum        of 2000 mg per day, given in divided doses twice daily

An oral solution of metformin has also been approved. It contains 500 mgof metformin hydrochloride per 5 mL and the following inactiveingredients: Saccharin Calcium, Potassium Bicarbonate, Xylitol,Hydrochloric Acid, Purified Water and Cherry Flavor

The maximum recommended daily dose is 2550 mg (25.5 mL) in adults and2000 mg (20 mL) in pediatric patients (10-16 years of age).

Provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the subject is also being        co-administered a therapeutically effective amount of a        multidrug and toxin extrusion transporter 1 (MATE1) substrate or        an organic cation transporter 2 (OCT2) substrate;    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme, and    -   wherein the therapeutically effective amount of the MATE1        substrate or OCT2 substrate, is reduced relative to a subject        who is not being administered levoketoconazole, or a        pharmaceutically acceptable salt thereof.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancersin a subject in need thereof, wherein the subject is beingco-administered a therapeutically effective amount of a multidrug andtoxin extrusion transporter 1 (MATE1) substrate or an organic cationtransporter 2 (OCT2) substrate, comprising:

-   -   reducing the amount of MATE1 substrate or OCT2 substrate being        administered to the subject, and    -   subsequently initiating administration of a therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme.

In some embodiments, reducing the amount of MATE1 substrate or OCT2substrate being administered to the subject comprises discontinuingadministration of the MATE1 substrate or OCT2 substrate.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the subject is also being        co-administered a therapeutically effective amount of metformin,        or a pharmaceutically acceptable salt thereof;    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme, and    -   wherein the therapeutically effective amount of metformin, or a        pharmaceutically acceptable salt thereof, is reduced relative to        a subject who is not being administered levoketoconazole, or a        pharmaceutically acceptable salt thereof.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancersin a subject in need thereof, wherein the subject is beingco-administered a therapeutically effective amount of metformin,comprising:

-   -   reducing the amount of metformin being administered to the        subject, and    -   subsequently initiating administration of a therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof, is determined via a titration scheme,        and    -   subsequently determining that the patient is to begin treatment        with a multidrug and toxin extrusion transporter 1 (MATE1)        substrate or an organic cation transporter 2 (OCT2) substrate,        wherein MATE1 substrate or OCT2 substrate, is administered in an        amount that is less than the amount that would be administered        to a patient who is not being administered levoketoconazole, or        a pharmaceutically acceptable salt thereof.

Also provided is a method of treating a disease chosen from Cushing'sdisease, syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof, is determined via a titration scheme,        and    -   subsequently determining that the patient is to begin treatment        with a multidrug and toxin extrusion transporter 1 (MATE1)        substrate or an organic cation transporter 2 (OCT2) substrate,        wherein MATE1 substrate or OCT2 substrate is administered at a        starting dose that is less than the amount that would be        administered to a patient who is not being administered        levoketoconazole, or a pharmaceutically acceptable salt thereof.

In some embodiments, the method further comprises increasing thestarting dose by an amount that is less than the amount that would beadministered to a patient who is not being administeredlevoketoconazole, or a pharmaceutically acceptable salt thereof. In someembodiments, the method further comprises increasing the starting doseby 250 mg increments as compared with the 500 mg increments that wouldbe administered to a patient who is not being administeredlevoketoconazole, or a pharmaceutically acceptable salt thereof.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof, wherein the therapeutically        effective amount of levoketoconazole, or a pharmaceutically        acceptable salt thereof, is determined via a titration scheme,        and    -   subsequently determining that the patient is to begin treatment        with metformin, wherein the metformin is administered in an        amount that is less than the amount that would be administered        to a patient who is not being administered levoketoconazole, or        a pharmaceutically acceptable salt thereof.

Also provided is a method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,in a subject in need thereof, wherein the subject is also beingadministered metformin, comprising:

-   -   administering a therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        to a subject in need thereof,    -   wherein the therapeutically effective amount of        levoketoconazole, or a pharmaceutically acceptable salt thereof,        is determined via a titration scheme, and    -   wherein the administration of levoketoconazole, or a        pharmaceutically acceptable salt thereof, increases the systemic        exposure to metformin by about 2-fold.

In certain embodiments, the disease is Cushing's syndrome. In certainembodiments, the disease is cyclic Cushing's syndrome. In certainembodiments, the disease is persistent or recurrent Cushing's syndrome.In certain embodiments, the subject has had previous surgery orradiation to treat the subject's Cushing syndrome. In certainembodiments, the subject has not had previous surgery or radiation totreat the subject's Cushing syndrome. In certain embodiments, thedisease is Cushing's disease.

In certain embodiments, the disease is exogenous hypercortisolism. Incertain embodiments, the disease is hypercortisolism. In certainembodiments, the disease is hyperglycemia. In certain embodiments, thedisease is multiple endocrine neoplasia type 1. In certain embodiments,the disease is McCune Albright syndrome. In certain embodiments, thedisease is Carney complex. In certain embodiments, the disease iscongenital adrenal hyperplasia. In certain embodiments, the disease isprecocious puberty.

In certain embodiments, the disease is a hormone-sensitive cancer. Incertain embodiments, the disease is prostate cancer and otherandrogen-sensitive cancers. In certain embodiments, the disease isbreast cancer, ovarian cancer or another cancer sensitive to estrogen orprogesterone.

In certain embodiments, the disease is a disorder susceptible oftreatment by levoketoconazole, or a pharmaceutically acceptable saltthereof.

In certain embodiments, the titration scheme comprises the up-titrationof the levoketoconazole, or a pharmaceutically acceptable salt thereofuntil one or more of the following conditions are met: (1) the subjecthas an adequate response; (2) the highest label-specified dose isreached; or (3) a dose limiting event occurs.

In certain embodiments, the titration scheme comprises administering afirst dose of the levoketoconazole, or a pharmaceutically acceptablesalt thereof, for a first time period, such as about one week;increasing the dose by an amount equal to an incremental value; anddetermining whether the subject tolerates the increased dose; whereinthe cycle is repeated so long as the subject tolerates the increaseddose, wherein the incremental value at each cycle repetition is the sameor different; and wherein if the subject does not tolerate the increaseddose, the dose for the patient is equal to the difference between thefurther increased dose and the incremental value for the last cyclerepetition. In certain embodiments, the initial dose oflevoketoconazole, or a pharmaceutically acceptable salt thereof, is 150mg twice daily. In certain embodiments, the incremental value is 150 mg.

In certain embodiments, the highest protocol-specified dose is 1200mg/day. In certain embodiments, the subject may receive a dose ofbetween 150 mg twice daily and to 600 mg twice daily. In certainembodiments, the dose is 150 mg once daily. In certain embodiments, thetherapeutically effective amount of the levoketoconazole, or apharmaceutically acceptable salt thereof, is between 150 mg and 1200 mgper day. In certain embodiments, the therapeutically effective amount ofthe levoketoconazole, or a pharmaceutically acceptable salt thereof, is150 mg once daily. In certain embodiments, the therapeutically effectiveamount of the levoketoconazole, or a pharmaceutically acceptable saltthereof, is 150 mg twice daily. In certain embodiments, thetherapeutically effective amount of the levoketoconazole, or apharmaceutically acceptable salt thereof, is 300 mg twice daily. Incertain embodiments, the therapeutically effective amount of thelevoketoconazole, or a pharmaceutically acceptable salt thereof, is 450mg twice daily. In certain embodiments, the therapeutically effectiveamount of the levoketoconazole, or a pharmaceutically acceptable saltthereof, is 600 mg twice daily.

In certain embodiments, the titration scheme comprises an initialreduction of dose. In certain embodiments, the initial dose oflevoketoconazole, or a pharmaceutically acceptable salt thereof, is 150mg twice daily and the reduced dose is 150 mg daily. In certainembodiments, after the initial reduction of dose, if the subjecttolerates the reduced dose, the dose is then increased via a titrationscheme as described herein. In certain embodiments, administration ismaintained at 150 mg daily.

In certain embodiments, the titration scheme comprises the up-titrationof the levoketoconazole, or a pharmaceutically acceptable salt thereofuntil the subject has an adequate response. In certain embodiments, anadequate response comprises normalization of urinary free cortisol (UFC)(e.g., 24-hour UFC, 4-hour UFC, 12-hour UFC, or other such UFCmeasurement) or late-night salivary cortisol (LNSC) or multiply-sampledserum cortisol (MSSC). In certain embodiments, UFC, LNSC or MSSCnormalization comprises at least a 50% decrease in mean UFC. In certainembodiments, an adequate response is less than a 50% decrease frombaseline cortisol levels. In certain embodiments, up-titration of thelevoketoconazole is continued until at least two of the cortisolmeasurements chosen from UFC, LNSC and MSSC are below baseline cortisollevels, such as an at least 50% decrease from the baseline cortisollevel. In certain embodiments, up-titration of the levoketoconazole isstopped when UFC, LNSC and MSSC are normalized or are below baselinecortisol levels. In certain embodiments, an adequate response comprisesan improvement in hypercortisolism, such as that measured via haircortisol or multiply-sampled sweat cortisol.

In certain embodiments, the titration scheme further comprises amaintenance phase wherein the subject is administered a therapeuticallyeffective amount of levoketoconazole at a fixed dose.

In certain embodiments, the titration scheme comprises the up-titrationof the levoketoconazole, or a pharmaceutically acceptable salt thereof,until the subject experiences a dose limiting event. In certainembodiments, after the subject experiences a dose limiting event, themethod further comprises reducing the dose of levoketoconazole, or apharmaceutically acceptable salt thereof. In certain embodiments, afterthe subject experiences a dose limiting event, the method furthercomprises temporarily interrupting administration of thelevoketoconazole, or a pharmaceutically acceptable salt thereof, untilthe dose limiting event is reversed and then resuming administration ofthe levoketoconazole, or a pharmaceutically acceptable salt thereof, atthe same or a reduced dose. In certain embodiments, administration ofthe levoketoconazole, or a pharmaceutically acceptable salt thereof, isnot resumed.

In certain embodiments, the dose limiting event is due to the increasedexposure to the MATE1 substrate or OCT2 substrate. In certainembodiments, the method further comprises informing the subject or amedical care worker that co-administration of the levoketoconazole, or apharmaceutically acceptable salt thereof, and the MATE1 substrate orOCT2 substrate may result in increased exposure to MATE1 substrate orOCT2 substrate. In certain embodiments, the method further comprisesinforming the subject or a medical care worker that co-administration ofthe levoketoconazole, or a pharmaceutically acceptable salt thereof, andMATE1 substrate or OCT2 substrate may result in one or moreexposure-related adverse reactions associated with administration of theMATE1 substrate or OCT2 substrate. In certain embodiments, the methodfurther comprises monitoring the serum concentration of the MATE1substrate or OCT2 substrate. In certain embodiments, the method furthercomprises monitoring the subject for one or more exposure-relatedadverse reactions associated with administration of the MATE1 substrateor OCT2 substrate.

In certain embodiments, the dose limiting event is due to the increasedexposure to metformin. In certain embodiments, the method furthercomprises informing the subject or a medical care worker thatco-administration of the levoketoconazole, or a pharmaceuticallyacceptable salt thereof, and metformin, or a pharmaceutically acceptablesalt thereof, may result in increased exposure to metformin, or apharmaceutically acceptable salt thereof. In certain embodiments, themethod further comprises informing the subject or a medical care workerthat co-administration of the levoketoconazole, or a pharmaceuticallyacceptable salt thereof, and metformin, or a pharmaceutically acceptablesalt thereof, may result in one or more exposure-related adversereactions associated with metformin administration. In certainembodiments, the method further comprises monitoring the serumconcentration of metformin, or a pharmaceutically acceptable saltthereof. In certain embodiments, the method further comprises monitoringthe subject for one or more exposure-related adverse reactionsassociated with metformin administration. These adverse reactions may bemild or moderate in severity. In certain embodiments, the method furthercomprises the one or more exposure-related adverse reactions are chosenfrom diarrhea, nausea/vomiting, flatulence, asthenia, indigestion,abdominal discomfort, lactic acidosis, and headache.

In certain embodiments, the dose limiting event is a QTc prolongationevent. In certain embodiments, the QTc prolongation event comprises atleast one QTc value representing greater than 60 msec increase frombaseline. In certain embodiments, the QTc prolongation event comprisesat least one confirmed QTc interval of greater than 470 msec, or incertain embodiments, at least one confirmed QTc interval of greater than500 msec. In certain embodiments, the QTc prolongation event comprisesan absolute QTc interval above 470 msec for males and above 480 msec forfemales. In certain embodiments, the QTc prolongation event comprises anabsolute QTc interval above 60 msec above the baseline.

In certain embodiments, the method further comprises monitoring for aneffect on the QTc interval.

In certain embodiments, the dose limiting event is an elevated liverfunction test (LFT). In certain embodiments, the LFT comprises testsanalyzing one of more of the following analytes in serum: alaninetransaminase (ALT), aspartate transaminase (AST), alkaline phosphatase(ALP), gamma glutamyl transferase (GGT), glutamate dehydrogenase (GLDH),lactate dehydrogenase (LDH) and/or bilirubin (unconjugated, conjugatedor total). In certain embodiments, the elevated LFT is at least 1.5times the upper limit of normal of the reference range. In certainembodiments, the elevated LFT is at least 2 times the upper limit ofnormal of the reference range. In certain embodiments, the elevationfurther comprises elevation of greater than one times the upper limit ofnormal of the reference range in either ALT or AST. To this effect, thenormal range for a given analyte can vary based on testing methodologyand from laboratory to laboratory. In certain embodiments, the methodfurther comprises monitoring liver function.

In certain embodiments, the dose limiting event is an increased risk ofType B lactic acidosis. In certain embodiments, the dose limiting eventis a blood pH less than 7.35 and lactate concentration of greater than 5mmol/L. In some embodiments, the subject also has a reduced serumbicarbonate concentration (e.g., less than 22 mmol/L) and/or an aniongap of greater than 12 meQ/L.

In certain embodiments, the dose limiting event is abnormal kidneyfunction. In certain embodiments, the method further comprisesmonitoring eGFR. In certain embodiments, abnormal kidney functioncomprises an estimated glomerular filtration rate (eGFR) of less than 30mL/min/1.73 m². In certain embodiments, abnormal kidney functioncomprises an eGFR of 30-45 mL/min/1.73 m². In certain embodiments, ifeGFR falls below 35 mL/min/1.73 m², the method further comprisesdiscontinuing administration of metformin.

In certain embodiments, the dose limiting event is a decreased fastingglucose level. In certain embodiments, the dose limiting event is a riskof further hypoglycemia.

In certain embodiments, the dose limiting event is anion gap acidosis.In certain embodiments, the dose limiting event is macrocytic anemiasecondary to low vitamin B-12.

In certain embodiments, the MATE1 substrate is chosen from thesubstrates (or pharmaceutically acceptable salts thereof) shown below:

MATE1 Substrate Associated Disease or Disorder Acyclovir A guanosineanalog used to treat herpes simplex, varicella zoster, herpes zoster.Cimetidine A histamine H2 receptor antagonist used to manage GERD,peptic ulcer disease, and indigestion. Ciprofloxacin Oral, intravenous,intratympanic, ophthalmic, and otic administration for a number ofbacterial infections Estrone Treatment of moderate to severe vasomotorsulfate symptoms associated with the monopause, and moderate to severesymptoms of vulval and vaginal atrophy associated with the menopause.Flecainide Prevent supraventricular arrhythmias, ventricular arrhythmiasand paroxysmal atrial fibrillation and flutter Ganciclovir A DNApolymerase inhibitor used to treat cytomegalovirus and herpetickeratitis of the eye. Guanidine For the reduction of the symptoms ofmuscle weakness and easy fatigability associated with the myasthenicsyndrome of Eaton-Lambert Levofloxacin Treatment of bacterialconjunctivitis caused by susceptible strains of the following organisms:Corynebacterium species, Staphylococus aureus, Staphylococcusepidermidis, Streptococcus pneumoniae, Streptococcus (Groups C/F/G),Viridans group streptococci, Acinetobacter Iwoffii, Haemophilusinfluenzae, Serratia marcescens Metformin A biguanide drug used inconjunction with diet and exercise for glycemic control in type 2diabetes mellitus and used off-label for insulin resistance inpolycystic ovary syndrome (PCOS). Nadolol Treat angina pectoris andhypertension Procainamide For the treatment of life-threateningventricular arrhythmias. Relebactam In combination with imipenem andcilastatin for the treatment of complicated urinary tract infections,including pyelonephritis, and complicated intra- abdominal infectionscaused by susceptible organisms Tipiracil In combination withtrifluridine, is indicated for the treatment of refractory mestastaticcolorectal cancer patients who keep progressing despite of treatmentwith standard chemotherapy and biologics Topotecan An antineoplasticagent used to treat ovarian cancer, small cell lung cancer, or cervicalcancer.In certain embodiments, the MATE1 substrate is chosen from cimetidine,abemacicilib, levofloxacin, ciprofloxacin, topotecan, metformin,cephalexin, acyclovir, cefradine, estrone sulfate, ganciclovir,guanidine, procainamide, and combinations thereof, or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the MATE1 substrate ismetformin, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the OCT2 substrate is chosen from the substrates(or pharmaceutically acceptable salts thereof) shown below:

OCT2 Substrate Associated Disease or Disorder Amantadine For thechemoprophylaxis, prophylaxis, and treatment of signs and symptoms ofinfection caused by various strains of influenza A virus. Also for thetreatment of parkinsonism and drug-induced extrapyramidal reactionsCholine Nutritional supplementation, also for treating dietary shortageor imbalance Choline Relief of pain and discomfort of common salicylatemouth ulcers, cold sores, denture sore spots, infant teething and mouthulcers, and sore spots due to orthodontic devices in children CimetidineTreatment and the management of acid-reflux disorders (GERD), pepticulcer disease, heartburn, and acid indigestion Cisplatin Treatment ofmetastatic testicular tumors, metastatic ovarian tumors and advancedbladder cancer Dalfampridine Neurofunctional modifier that helps improvewalking speed in patients with multiple sclerosis Dofetilide Maintenanceof normal sinus rhythm (delay in time to recurrence of atrialfibrillation/atrial flutter Dopamine Correction of hemodynamicimbalances present in the shock syndrome due to Myocardial infarction,trauma, endotoxic septicemia, open-heart surgery, renal failure, andchronic cardiac decompensation as in congestive failure EpinephrineEmergency treatment of allergic reactions (Type I) Guanfacine Treat ADHDHistamine Diagnostic aid for evaluation of gastric acid secretoryfunction Lamivudine Treatment of HIV infection and chronic hepatitis B(HBV). Linagliptin Treatment of type II diabetes Memantine Managemoderate to severe Alzheimer's dementia Metformin Adjunct to diet andexercise to increase glycemic control in patients diagnosed with type 2diabetes mellitus Norepinephrine Treat patients in vasodilatory shockstates such as septic shock and neurogenic shock Oxaliplatin Treatmentof advanced carcinoma of the colon or rectum and for adjuvant treatmentof stage III colon cancer patients Pramipexole Symptomatic treatment ofParkinson's disease Ranitidine Treatment of short-term treatment ofactive duodenal ulcer, treating gastric acid hypersecretion due toZollinger-Ellison syndrome, systemic mastocytosis, and other conditionsthat may pathologically raise gastric acid levels Reserpine Treatment ofhypertension Tipiracil Treatment of refractory mestastatic colorectalcancer patients Varenicline Aid in smoking cessation

In certain embodiments, the OCT2 substrate is chosen from amantadine,amiloride, cimetidine, dopamine, famotidine, memantine, metformin,pindolol, procainamide, ranitidine, varenicline, and oxaliplatin, or apharmaceutically acceptable salt thereof. In certain embodiments, theOCT2 substrate is metformin, or a pharmaceutically acceptable saltthereof.

In certain embodiments, the dose of the MATE1 substrate or OCT2substrate is decreased. In certain embodiments, the dose of the MATE1substrate or OCT2 substrate is reduced by at least 5%, such as by atleast 10%, by at least 20%, by at least 25%, by at least 30%, by atleast 40%, by at least 50%, by at least 60%, by at least 70%, by atleast 75%, by at least 80%, or by at least 90%. In certain embodiments,the frequency of administration of the MATE1 substrate or OCT2 substrateis decreased. For example, when the dose is not reduced, the frequencyof administration might be extended from twice daily (BID) to once daily(QD), or to every other day (QOD), and on.

In certain embodiments, the dose of the metformin, or a pharmaceuticallyacceptable salt thereof, is decreased. In certain embodiments, the doseof the metformin, or a pharmaceutically acceptable salt thereof, isreduced by at least 5%, such as by at least 10%, by at least 20%, by atleast 25%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 75%, by at least 80%, or by atleast 90%. In certain embodiments, the frequency of administration ofthe metformin, or a pharmaceutically acceptable salt thereof, isdecreased. For example, when the dose is not reduced, the frequency ofadministration might be extended from twice daily (BID) to once daily(QD), or to every other day (QOD), and on.

While the disclosed compounds may be administered as the raw chemical,it is also possible to present them as a pharmaceutical formulation.Accordingly, provided herein are pharmaceutical formulations whichcomprise one or more of certain compounds disclosed herein, or one ormore pharmaceutically acceptable salts thereof, together with one ormore pharmaceutically acceptable carriers thereof and optionally one ormore other therapeutic ingredients. The carrier(s) must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art. The pharmaceutical compositionsdisclosed herein may be manufactured in any manner known in the art,e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orcompression processes.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compounddisclosed herein or a pharmaceutically acceptable salt thereof (“activeingredient”) with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated to provide slow or controlledrelease of the active ingredient therein. All formulations for oraladministration should be in dosages suitable for such administration.The push-fit capsules can contain the active ingredients in admixturewith filler such as lactose, binders such as starches, and/or lubricantssuch as talc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

In some embodiments, the dosage form is suitable for oral administrationand contains one or more pharmaceutical excipients. In some embodiments,the unit dosage form is an immediate release tablet comprising 150 mglevoketoconazole together with microcrystalline cellulose, lactose, cornstarch, colloidal silicon dioxide, and magnesium stearate. In someembodiments, the tablet is film coated.

Compounds may be administered orally or via injection at a dose of from0.1 to 500 20 mg/kg per day. The dose range for adult humans isgenerally from 150 mg to 1.2 g/day. Tablets or other forms ofpresentation provided in discrete units may conveniently contain anamount of one or more compounds which is effective at such dosage or asa multiple of the same, for instance, units containing 150 mg to 1200mg.

Examples of embodiments of the present disclosure are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using thedisclosure. The examples are not intended in any way to otherwise limitthe scope of the disclosure.

EXAMPLE Example 1—Drug-Drug Interaction Study with MATE1 and OCT2Substrates

A study was designed to evaluate levoketoconazole as an inhibitor ofMATE1 or OCT2. Compounds that are substrates or inhibitors of thetransporters may be victims or perpetrators in drug-drug interactions.Experiments were carried out as described in the FDA and EMA draftguidance documents for Drug Interaction Studies (FDA 2017, EMA 2013).The probe substrate was [¹⁴C]-metformin.

Short-term stability testing was performed on the test articlesolutions. Dose solutions at the low and high test articleconcentrations (e.g., 0.03 and 50 μM) were prepared in each incubationmedia and stored in the selected vessel type at room temperature and37±2° C. for 6 hours. After the applicable storage duration, solutionsat the same test article concentrations were re-prepared and analyzedwith the previously stored samples to determine stability. Solutionswere stable if the difference between mean responses (e.g., area ratios)for fresh and stored samples was within ±15% for dosing solutions.

The toxicity of the test articles to the various cell system in thestudy were assessed separately by measuring the lactate dehydrogenase(LDH) released from the cells. Incubation media not exposed to cellswill serve as a background control. Incubation media were collected fromcells exposed to no test article solvent control incubation media(containing only positive control substrate and inhibitor solvent, 0.2%v/v DMSO) (negative control), 1% Triton X-100 (positive control),solvent control (0 μM sample containing 0.2% v/v DMSO and test articlesolvent) and the test articles at select concentrations. Toxicityobserved with the test articles was ≤25% compared to that of thepositive control.

Stock solutions of the radiolabeled transporter substrates (e.g., 10 mM)were prepared in DMSO. [¹⁴C]-Metformin (1 mM) was provided as a solidand was prepared in Hank's Balanced Salt Solution (HBSS). Controlinhibitors were prepared in DMSO (e.g., 10 mM). The substrates and thecontrol inhibitors or DMSO for the solvent control were spiked intoincubation media in 0.1% v/v DMSO. The test article was spiked into theincubation media in an appropriate solvent and the solvent concentrationwas adjusted so the solvent was the same in all incubations.

The non-specific binding of the test articles to select incubationvessels absent cells was evaluated. The test articles were mixed withapplicable incubation media, separately, at low and high concentrationsand incubated in 24-well cell culture plates but absent cells. At theend the incubation period, aliquots of mixture were collected andanalyzed by LC-MS/MS and compared to the dose solution (100% solution).A standard curve was be included. Recovery was determined from arearatio.

Before the experiment, cell culture plates (transporter-expressing andcontrol cells) were removed from the incubator, the cell culture mediumwas removed and incubation medium (1 mL) was added to the plate to rinsethe cell culture medium from the cells. Incubation medium was replacedwith incubation medium containing levoketoconazole, positive controlinhibitor or solvent control (0.3 mL) and the plates were preincubated.After preincubation, incubation medium was replaced with incubationmedium containing levoketoconazole, positive control inhibitor orsolvent control and the probe substrate. Samples were incubated for thedesignated time. After incubation, incubation medium was removed, andcells were rinsed once with 1 mL of ice-cold phosphate-buffered saline(PBS) containing 0.2% w/v bovine specific antigen (BSA) and twice withice-cold PBS. The PBS was removed, and 0.5 mL of sodium hydroxide (0.1M) was added and pipetted up and down to dissolve and suspend the cells.An aliquot of the medium was added to a 96-well plate, diluted withscintillation fluid and analyzed on a MicroBeta scintillation counter.The amount of protein in each incubation was determined by bicinchoninicacid analysis.

The uptake of the relevant probe substrate in transporter expressingcells and control cells in the presence and absence of a known inhibitorwas the positive control. The transporter specific uptake of the probesubstrate or the test article was determined by subtracting the uptakein the control cells from the uptake in the transporter expressingcells. IC₅₀ values were determined from the decrease in activity (e.g.,percent of control) when inhibition exceeds 50% and calculated bynon-linear regression with the four-parameter IC₅₀ equation. Tocalculate recovery, samples were taken from the incubation media at zerominutes (dose solution) and the final incubation time and calculated.

Human embryonic kidney 293 (HEK293) cells expressing transportertransfected with vectors containing human transporter cDNA for MATE1,OCT2, and control cells (HEK293 cells transfected with only vector) wereused in experiments to evaluate levoketoconazole as an inhibitor ofMATE1 or OCT2.

HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)supplemented with fetal bovine serum (FBS, 8.9% v/v),antibiotic/antimycotic (0.89% v/v) and L-glutamine (1.79 mM) in ahumidified culture chamber (37±1° C., 95±5% relative humidity, and 5±1%CO₂) in cell culture flasks. The medium was replaced every 2 to 3 days,and the cells were passaged when they became confluent. HEPES(Sigma-Aldrich, Saint Louis) was the incubation medium forOCT2-expressing HEK293 cells. Cell loss was not observed in incubationswith OCT2-expressing HEK293 cells.

Caco-2 cells were cultured on a porous membrane in a transwell plate andallowed to form a confluent monolayer with tight junctions. Themonolayer separates the apical and basolateral compartments of thetranswell. Caco-2 cells were cultured in Eagle's minimal essentialmedium (EMEM) supplemented with FBS (8.9% v/v), non-essential aminoacids (0.89% v/v) and penicillin-streptomycin (45 U/mL and 45 μg/mL,respectively) in a humidified culture chamber (37±1° C., 95±5% relativehumidity, and 5±1% CO₂). The medium was replaced every 2 to 3 days, andthe cells were passaged when they became confluent.

MDCKII cells were cultured on a porous membrane in a transwell plate andallowed to form a confluent monolayer with tight junctions. Themonolayer separates the apical and basolateral compartments of thetranswell. MDCKII cells were cultured in DMEM supplemented with FBS (10%v/v) and penicillin-streptomycin (45 U/mL and 45 μg/mL, respectively) ina humidified culture chamber (37±2° C., 95±5% relative humidity, and5±1% CO₂) in cell culture flasks. The medium was replaced every 2 to 3days, and the cells were passaged when they became confluent.

The tables below show the results of the inhibition experiments withketoconazole and levoketoconazole. Where applicable, n is the number ofreplicates, NA is Not applicable, and SD refers to the standarddeviation. Unless otherwise noted, values are triplicate determinationsrounded to three significant figures with standard deviations rounded tothe same degree of accuracy. Percentages are rounded to one decimalplace except percentages ≥100, which are rounded to the nearest wholenumber.

TABLE 1 Ketoconazole OCT2 inhibition in HEK293 cells using [¹⁴C]-metformin (10 μM) for the probe substrate Background corrected[Inhibitor] Uptake (pmol/mg) uptake rate % IC₅₀ Inhibitor (μM) ControlOCT2 (pmol/mg/min) control parameters Solvent 0 5.83 ± 243 ± 22 119 100IC₅₀: 1.52 μM control 0.56 Slope: 1.41 Keto- 0.03 4.25 ± 190 ± 7  92.878.2 Min: 0% conazole 0.13 Max: 92.0% 0.1 3.76 ± 241 ± 34 119 100 0.580.3 2.71 ± 219 ± 25 108 91.2 0.90 1 3.38 ± 127 ± 23 62.1 52.3 2.37 31.86 (n = 70.0 ± 8.9 34.1 28.7 2) 10 2.33 ± 22.1 ± 3.1 9.87 8.3 1.02 301.54 ± 7.27 ± 2.86 2.4 0.96 1.82

TABLE 2 Levoketoconazole OCT2 inhibition in HEK293 cells using[¹⁴C]-metformin (10 μM) for the probe substrate Background corrected[Inhibitor] Uptake (pmol/mg) uptake rate % IC₅₀ Inhibitor (μM) ControlOCT2 (pmol/mg/min) control parameters Solvent 0 4.14 ± 0.16 286 ± 23 141 100 IC₅₀: 0.218 control μM Levoketo- 0.03 5.10 ± 0.74 318 ± 46  156111 Slope: conazole 0.1 4.31 ± 0.33 247 ± 13  121 86.2 0.970 0.3 3.35 ±0.64 155 ± 11  75.9 53.9 Min: 0% 1 2.26 ± 0.52 69.6 ± 7.7  33.7 23.9Max: 127% 3 1.95 ± 0.93 28.2 ± 6.0  13.1 9.3 10 1.88 ± 0.66 7.71 ± 1.342.91 2.1 30 1.61 ± 0.45 3.44 ± 2.05 0.915 0.6

TABLE 3 Ketoconazole MATE1 inhibition in HEK293 cells using[¹⁴C]-metformin (10 μM) for the probe substrate Background Uptakecorrected (pmol/mg uptake [In- protein) rate IC₅₀ hibitor] (Average ±SD) (pmol/ % pa- Inhibitor (μM) Control MATE1 mg/min) Control rametersNo 0 6.17 ± 508 ± 38 100 NA NA Solvent 1.43 Control Solvent 0 5.27 ± 540± 33 107 100 IC₅₀: Control 1.26 0.279 μM Keto- 0.03 6.53 ± 453 ± 29 89.383.6 Slope: conazole 1.52 1.06 0.1 3.90 ± 382 ± 27 75.6 70.8 Min: 0%2.13 Max: 0.3 4.58 ± 235 ± 8  46.1 43.1 92.0% 1.36 1 3.06 ± 104 ± 2 20.2 18.9 1.56 3 1.90 ± 44.2 ± 8.46 7.9 0.81 2.2 10 2.48 ± 16.4 ± 2.792.6 0.09 0.6 25 1.90 ± 12.7 ± 2.17 2.0 0.51 1.5

TABLE 4 Levoketoconazole MATE1 inhibition in HEK293 cells using[¹⁴C]-metformin (10 μM) for the probe substrate Uptake Background(pmol/mg protein) corrected uptake [Inhibitor] (Average ± SD) rate %IC₅₀ Inhibitor (μM) Control MATE1 (pmol/mg/min) Control parameters NoSolvent 0 5.66 ±  433 ± 18 85.5 NA NA Control 1.56 Solvent 0 5.38 ±  437± 25 86.4 100 IC₅₀: 0.248 Control 0.55 μM Levoketo- 0.03 5.25 ±  400 ±18 79.0 91.4 Slope: 0.941 conazole 0.96 Min: 0% 0.1 4.21 ±  316 ± 3062.3 72.1 Max: 104% 0.74 0.3 3.58 ±  210 ± 11 41.4 47.9 1.56 1 3.81 ±101 ± 9 19.4 22.5 0.16 3 2.64 (n = 44.0 ± 8.28 9.6 2) 5.6  10 1.82 (n =13.6 ± 2.36 2.7 2) 0.9  25 2.82 ± 7.72 ± 0.981 1.1 1.22 1.09

As can be seen from the foregoing tables, levoketoconazole inhibitedOCT2 with an IC₅₀ of 0.218 μM, which was more potent than racemicketoconazole (IC₅₀=1.52 μM).

Example 2—Phase I Study of Levoketoconazole and MetforminCoadministration

This was a Phase I, open-label, 3-period, fixed-sequence study in 32healthy male and female subjects (about 16 per sex) designed to evaluatethe effect of levoketoconazole on the pharmacokinetics of a single 500mg dose of metformin. The study consisted of a screening period of up to21 days, a metformin-only treatment period (Period 1, Treatment A), alevoketoconazole dose escalation treatment period (Period 2) to achievethe dose level to be used in Period 3, and a metformin andlevoketoconazole co-administration treatment period (Period 3, TreatmentB). Urine was also collected prior to dose administration and inspecified intervals (0-6 hours, 6-12 hours, 12-24 hours, 24-36 hours,and 36-48 hours post-dose) for 48 hours after dose administration tomeasure the recovery of metformin.

All subjects received a single oral dose of 500 mg metformin in Period1.

Period 2 was a dose escalation period designed to increase thelevoketoconazole dose stepwise to the levoketoconazole dose level of 600mg taken every 12 hours (Q12H) used in Period 3. Period 2 lasted fromDay 4 to 27 of the study. Subjects received 150 mg, 300 mg, 450 mg, and600 mg levoketoconazole Q12H (approximate timing when at home) as fourconsecutive weekly escalation cycles, starting on Day 4 with doselevel 1. For dose level 1, subjects received 150 mg Q12H. For doselevels 2 and 3, subjects received 300 mg and 450 mg levoketoconazoleQ12H, respectively. For dose level 4, subjects received 600 mglevoketoconazole Q12H. Subjects received 6 doses of 600 mglevoketoconazole alone before co-administration of levoketoconazole andmetformin in Period 3.

Period 3 was the drug-drug interactions assessment period in whichlevoketoconazole 600 mg was co-administered with a single oral dose of500 mg metformin on the morning of Day 28. Subjects received fiverepeated oral doses of 600 mg levoketoconazole Q12H for a total dailydose of 1200 mg. Period 3 lasted from day 28 to 31 of the study. Again,urine was collected prior to dose administration and in specifiedintervals (0-6 hours, 6-12 hours, 12-24 hours, 24-36 hours, and 36-48hours post-dose) for 48 hours after dose administration to measure therecovery of metformin.

The preliminary metformin pharmacokinetic parameters are described inTable 5. There is an increase in C max and AUC, as well as a decrease inCl/F in Period 3.

TABLE 5 Cmax Tmax AUClast Tlast AUCINF Vz/F Cl/F t1/2 Period (ng/mL) (h)(h*ng/mL) (h) (h*ng/mL) (L) (L/h) (h) 1 N 30 30 30 30 19 19 19 19 Median822 4.00 6450 48.0 6270 1350 79.7 12.1 Geometric 844 NC 6280 NC 60901340 82.2 11.3 Mean CV % 20.8 NC 17.7 NC 14.2 66.7 14.2 55.00 GeometricMean 3 N 17 17 17 17 16 16 16 16 Median 1570 4.00 14600 48.0 15100 52133.1 10.7 Geometric 1520 NC 13800 NC 14000 547 35.7 10.6 Mean CV % 19.4NC 24.2 NC 25.1 37.7 25.1 23.5 Geometric Mean

A comparison of Metformin co-administered with levoketoconazole inPeriod 3 vs. metformin alone in Period 1 was undertaken. A summary ofthe levoketoconazole effect on the pharmacokinetics of metformin isshown in Table 6. Administration of levoketoconazole increases thesystemic exposure to metformin by about 2-fold.

TABLE 6 Geometric Geometric 90% LSM LSM Test/Reference ConfidenceParameter Units N n (Test) (Reference) (%) Interval AUCinf h*ng/mL 17 3013900 6330 2.2 (2.03, 2.39) AUClast h*ng/mL 17 30 13800 6270 2.19 (2.02,2.37) CLr L/h 17 30 9.61 25.6 0.375 (0.308, 0.456) Cmax ng/mL 17 30 1530844 1.81 (1.67, 1.96)

A comparison of urine pharmacokinetic parameters from Metforminco-administered with levoketoconazole in Period 3 vs. metformin alone inPeriod 1 was undertaken, with the results shown in Table 7.

TABLE 7 Treatment Ae Fe CLr Period (ug) (%) (L/h) Period 1 161000 32.225.6 (n = 30) (34.7) (34.7) (31.5) [142000, 182000] [28.4, 36.5] [22.9,28.8] Period 3 132000 26.3 9.54 (n = 17) (57.7) (57.7) (65.5) [99900,173000] [20.0, 34.7] [7.02, 13.0]

The plasma data and the additional pharmacokinetics parameters fromplasma support a drug drug interaction between levoketoconazole andmetformin via a mechanism of decreased total body clearance ofmetformin. The additional urine data support an effect oflevoketoconazole to reduce urine clearance of metformin. Metformin isnot metabolized and is excreted unchanged primarily if not exclusivelyin urine.

These previously undescribed effects of levoketoconazole to inhibit OCT2in vitro, coupled with novel effect of co-administration of the drugs onmetformin renal and total body clearances and urine excretion, and themagnitude of the increase in metformin AUC/C max that results frominhibition of clearance are unpredictable and clinically significant.

The various embodiments described above can be combined to providefurther embodiments. All the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary, to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. (canceled)
 2. (canceled)
 3. A method of treating a disease chosenfrom Cushing's disease, Cushing's syndrome, cyclic Cushing's syndrome,exogenous hypercortisolism, hypercortisolism, hyperglycemia, multipleendocrine neoplasia type 1, McCune Albright syndrome, Carney complex,congenital adrenal hyperplasia, precocious puberty, andhormone-sensitive cancers, comprising: administering a therapeuticallyeffective amount of levoketoconazole, or a pharmaceutically acceptablesalt thereof, to a subject in need thereof, wherein the subject is alsobeing co-administered a therapeutically effective amount of metformin,or a pharmaceutically acceptable salt thereof; wherein thetherapeutically effective amount of levoketoconazole, or apharmaceutically acceptable salt thereof, is determined via a titrationscheme, and wherein the therapeutically effective amount of metformin,or a pharmaceutically acceptable salt thereof, is reduced relative to asubject who is not being administered levoketoconazole, or apharmaceutically acceptable salt thereof.
 4. A method of treating adisease chosen from Cushing's disease, Cushing's syndrome, cyclicCushing's syndrome, exogenous hypercortisolism, hypercortisolism,hyperglycemia, multiple endocrine neoplasia type 1, McCune Albrightsyndrome, Carney complex, congenital adrenal hyperplasia, precociouspuberty, and hormone-sensitive cancers in a subject in need thereof,wherein the subject is being co-administered a therapeutically effectiveamount of metformin, comprising: reducing the amount of metformin beingadministered to the subject, and subsequently initiating administrationof a therapeutically effective amount of levoketoconazole, or apharmaceutically acceptable salt thereof wherein the therapeuticallyeffective amount of levoketoconazole, or a pharmaceutically acceptablesalt thereof, is determined via a titration scheme.
 5. (canceled) 6.(canceled)
 7. A method of treating a disease chosen from Cushing'sdisease, Cushing's syndrome, cyclic Cushing's syndrome, exogenoushypercortisolism, hypercortisolism, hyperglycemia, multiple endocrineneoplasia type 1, McCune Albright syndrome, Carney complex, congenitaladrenal hyperplasia, precocious puberty, and hormone-sensitive cancers,comprising: administering a therapeutically effective amount oflevoketoconazole, or a pharmaceutically acceptable salt thereof, to asubject in need thereof, wherein the therapeutically effective amount oflevoketoconazole, or a pharmaceutically acceptable salt thereof, isdetermined via a titration scheme, and subsequently determining that thepatient is to begin treatment metformin, wherein metformin isadministered in an amount that is less than the amount that would beadministered to a patient who is not being administeredlevoketoconazole, or a pharmaceutically acceptable salt thereof.
 8. Amethod of treating a disease chosen from Cushing's disease, syndrome,cyclic Cushing's syndrome, exogenous hypercortisolism, hypercortisolism,hyperglycemia, multiple endocrine neoplasia type 1, McCune Albrightsyndrome, Carney complex, congenital adrenal hyperplasia, precociouspuberty, and hormone-sensitive cancers, comprising: administering atherapeutically effective amount of levoketoconazole, or apharmaceutically acceptable salt thereof, to a subject in need thereof,wherein the therapeutically effective amount of levoketoconazole, or apharmaceutically acceptable salt thereof, is determined via a titrationscheme, and subsequently determining that the patient is to begintreatment with metformin, wherein metformin is administered at astarting dose that is less than the amount that would be administered toa patient who is not being administered levoketoconazole, or apharmaceutically acceptable salt thereof.
 9. (canceled)
 10. The methodof claim 3, wherein the disease is chosen from Cushing's syndrome andcyclic Cushing's syndrome.
 11. The method of claim 10, wherein thedisease is persistent or recurrent Cushing's syndrome.
 12. The method ofclaim 10, wherein the subject has had previous surgery or radiation totreat the subject's Cushing syndrome.
 13. The method of claim 10,wherein the subject has not had previous surgery or radiation to treatthe subject's Cushing syndrome.
 14. The method of claim 3, wherein thedose of the metformin, or a pharmaceutically acceptable salt thereof, isdecreased.
 15. The method of claim 14, wherein the dose of themetformin, or a pharmaceutically acceptable salt thereof, is reduced byat least 25%.
 16. The method of claim 14, wherein the dose of themetformin, or a pharmaceutically acceptable salt thereof, is reduced byat least 50%.
 17. The method of claim 3, wherein the frequency ofadministration of the metformin, or a pharmaceutically acceptable saltthereof, is decreased.
 18. The method of claim 3, wherein the titrationscheme comprises the up-titration of the levoketoconazole, or apharmaceutically acceptable salt thereof until one or more of thefollowing conditions are met: (1) the subject has an adequate response;(2) the highest label-specified dose is reached; or (3) a dose limitingevent occurs.
 19. The method of claim 3, wherein the titration schemecomprises administering a first dose of the of the levoketoconazole, ora pharmaceutically acceptable salt thereof, for a first time period;increasing the dose by an amount equal to an incremental value; anddetermining whether the subject tolerates the increased dose; whereinthe cycle is repeated so long as the subject tolerates the increaseddose, wherein the incremental value at each cycle repetition is the sameor different; and wherein if the subject does not tolerate the increaseddose, the dose for the patient is equal to the difference between thefurther increased dose and the incremental value for the last cyclerepetition.
 20. The method of claim 19, wherein the first dose of thelevoketoconazole, or a pharmaceutically acceptable salt thereof, is 150mg administered twice daily.
 21. The method of claim 19, wherein theincremental value is 150 mg per day.
 22. The method of claim 3, whereinthe therapeutically effective amount of the levoketoconazole, or apharmaceutically acceptable salt thereof, is between 150 mg and 1200 mgper day.
 23. The method of claim 3, wherein the therapeuticallyeffective amount of the levoketoconazole, or a pharmaceuticallyacceptable salt thereof, is 600 mg twice daily.
 24. The method of claim3, further comprising informing the subject or a medical care workerthat co-administration of the levoketoconazole, or a pharmaceuticallyacceptable salt thereof, and the metformin, or a pharmaceuticallyacceptable salt thereof, may result in increased exposure to metformin.25. The method of claim 3, further comprising informing the subject or amedical care worker that co-administration of the levoketoconazole, or apharmaceutically acceptable salt thereof, and metformin, or apharmaceutically acceptable salt thereof, may result in one or moreexposure-related adverse reactions associated with metformin, or apharmaceutically acceptable salt thereof, administration.
 26. The methodof claim 3, further comprising monitoring the serum concentration ofmetformin, or a pharmaceutically acceptable salt thereof.
 27. The methodof claim 3, further comprising monitoring the subject for one or moreexposure-related adverse reactions associated with metformin, or apharmaceutically acceptable salt thereof, administration.
 28. The methodof claim 27, wherein the one or more exposure-related adverse reactionsare chosen from diarrhea, nausea/vomiting, flatulence, asthenia,indigestion, abdominal discomfort, lactic acidosis, and headache.